The Chlamydiaceae are pathogens of humans, livestock, companion animals and many species of wildlife. The global impact of chlamydial diseases is significant. Mice are quite commonly used to model human infection and there are several strengths in regard to the mouse model. There is new information emerging that there are multiple variants of C. muridarum within the populations of our present available strains. Yet we know little of the extent of C. muridarum adaptation over the past 60 years and untold numbers of passages in hen's eggs and then in cell culture since it was originally isolated. Were fresh and more varied isolates defined and a broader repertoire of rodent-adapted chlamydiae available to investigators, better modeling of human chlamydial infections and immunity could be realized. Isolation of novel rodent chlamydiae could also improve our understanding of chlamydial variant stability or variation in vitro and in vivo as well as chlamydial population dynamics in various hosts. We present serological and molecular evidence that rodent chlamydiae circulates in Peromyscus spp. mice in nature and in some domesticated Peromyscus spp laboratory mice. In this proposal, we hypothesize that one can successfully isolate novel rodent chlamydiae from these sources. We will test this hypothesis in 2 Aims. In Aim 1 (Ramsey, Midwestern University), we will trap Peromyscus spp. mice and screen these for chlamydial nucleic acids by polymerase chain reaction (PCR). Mice will be euthanized and those mice that are PCR-positive will be selected for further investigation. In Aim 2, positive tissue samples selected in Aim 1 will be shipped to the Regional Biocontainment Laboratory at the Univ. Tenn. Health Sciences Center (Peters, UTHSC) where they will be inoculated into highly chlamydia-susceptible DBA mice under ABSL3 conditions. Tissues from PCR-positive domesticated Peromyscus mice will also be used to inoculate DBA mice. Replicating the original methods used to isolate C. muridarum, we will conduct serial passage in mice to amplify the pathogen. Chlamydial isolates will be expanded in culture with maintenance at low in vitro passage number. Isolates will be initially characterized by phenotypic growth in culture (e.g., growth rate, plaque assay/efficiency, and micromorphometirc analysis of inclusions). The genome of cloned candidate isolates (up to 10) will be sequenced. These in vitro studies of isolates will set the stage for future ones involving establishing in vivo phenotypes in laboratory mice. Further work could characterize variant population dynamics in vivo and in vitro. We believe the aims and scope of this proposal constitute an exploratory study that is potential of high reward in that they will lay the ground work for extending studies in new directions by identifying new agents that will improve modeling of human chlamydial infections, provide insight into chlamydial genomics, population dynamics, evolution and adaptation, and host- chlamydia interactions.